Functional Analysis of a Unique Troponin C Mutation, GLY159ASP, that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle

Abstract

Background-Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca 2ϩ sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca 2ϩ -regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation. Methods and Results-Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca 2ϩ -activated force was similar in cTnC G159D and donor myocytes, but the Ca 2ϩ sensitivity of cTnC G159D myocytes was higher (EC 50 G159D/donorϭ0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca 2ϩ sensitivity (EC 50 G159D/donorϭ0.55Ϯ0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca 2ϩ sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca 2ϩ sensitivity was increased (EC 50 P/unPϭ4.7Ϯ1.9) but not with cTnC G159D troponin (EC 50 P/unPϭ1.2Ϯ0.1). Conclusions-We propose that uncoupling of the relationship between phosphorylation and Ca 2ϩ sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation. (Circ Heart Fail. 2009;2:456-464.) Key Words: cardiomyopathy Ⅲ contractility Ⅲ heart failure D ilated cardiomyopathy (DCM) is a common cause of sudden death and heart failure, and it is estimated that 20% to 30% of cases of DCM are caused by mutations in specific proteins. 1 Many cases of inherited "pure" DCM that are not associated with other symptoms, such as conduction disease, are caused by mutations in contractile proteins, including actin, myosin, tropomyosin, and all 3 subunits of troponin. 1,2 These cardiomyopathy-causing mutations present a unique opportunity to link genotype with phenotype. Because mutations at different sites in several different contractile proteins can produce a single phenotype, it has been proposed that mutations causing the same phenotype alter the contractile mechanism in the same way. In support of this hypothesis, a number of DCM mutations have been shown to cause decreased Ca 2ϩ sensitivity linked to reduced troponin C Ca 2ϩ -binding affinity and decreased cross-bridge turnover rate in vitro. [3][4][5][6][7][8][9] However, recent work, studying both recombinant proteins and intact myofibrils, has provided results that contradict the simple hypothesis and this raises doubts about the physiological relev...

Molecular Mechanism of the E99K Mutation in Cardiac Actin (ACTC Gene) That Causes Apical Hypertrophy in Man and Mouse

“…In addition, we have demonstrated that the Ca 2ϩ sensitivity of E99K actin-containing thin filaments was uncoupled from the PKA-dependent phosphorylation of troponin I. Uncoupling has been reported with other HCM-or DCM-causing mutations in thin filament proteins (16,28,46), and in DCM it seems to be the only consistent effect of disease-causing mutations (21). In contrast, HCM mutations are always associated with higher myofibrillar Ca 2ϩ sensitivity.…”

“…Uncoupling was previously reported in the TNNTI3 R145G mutation (46) and the TNNC1 L29Q mutation (40) that cause HCM but also in DCM mutations (ACTC E361G, TNNC1 G149D, and TPM1 E40K (16,21,28)) and in troponin from human myectomy samples (56). Thus, it appears that uncoupling may be a default consequence of structural perturbations.…”

“…Cardiac actin is the only contractile protein that has an identical amino acid sequence in humans and mice as well as an identical isoform distribution (80% cardiac actin, ACTC and 20% skeletal actin). This is an important property because we have found that the effect of HCM and DCM mutations on function in vitro can vary significantly depending on species, isoform, and posttranslational modifications (16,22,28). Actin transgenes with the MHC promoter lead to substitution of actin rather than overexpression (27), and we observed that myofibrillar protein content is normal (Fig.…”

“…It has been widely supposed that mutant protein would be expressed in the heart at up to 50% of total protein and that it exerts its pathological effects by acting as a poison peptide that alters normal function. Direct measurements in human heart tissue have confirmed the poison peptide hypothesis in a few cases (15)(16)(17). The challenge is to explain how over 600 different mutations can cause the same phenotype.…”

“…Moreover, measurements are usually made in heterologous systems with the mutation introduced into mouse or human isoforms of a protein and then tested in a system containing partner proteins from a different species. Recent studies have shown that species, isoform, and post-translational modification context can have a significant influence on the functional effect of a mutation (16,22,23).…”